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- PDB-9bge: Cryo-EM structure of mAb8-24 bound to 426c.WITO.TM.SOSIP -

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Basic information

Entry
Database: PDB / ID: 9bge
TitleCryo-EM structure of mAb8-24 bound to 426c.WITO.TM.SOSIP
Components
  • (Fv domain of ...) x 2
  • 426c.WITO.TM.SOSIP
KeywordsIMMUNE SYSTEM / HIV / antibody
Biological speciesHuman immunodeficiency virus 1
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsHurlburt, N.K. / Pancera, M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01 AI104384 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI174242-01 United States
CitationJournal: J Virol / Year: 2024
Title: Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env.
Authors: Parul Agrawal / Maria L Knudsen / Anna MacCamy / Nicholas K Hurlburt / Arineh Khechaduri / Kelsey R Salladay / Gargi M Kher / Latha Kallur Siddaramaiah / Andrew B Stuart / Ilja Bontjer / ...Authors: Parul Agrawal / Maria L Knudsen / Anna MacCamy / Nicholas K Hurlburt / Arineh Khechaduri / Kelsey R Salladay / Gargi M Kher / Latha Kallur Siddaramaiah / Andrew B Stuart / Ilja Bontjer / Xiaoying Shen / David Montefiori / Harry B Gristick / Pamela J Bjorkman / Rogier W Sanders / Marie Pancera / Leonidas Stamatatos /
Abstract: VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and ...VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies.
IMPORTANCE: HIV-1 broadly neutralizing antibodies will be a key component of an effective HIV-1 vaccine, as they prevent viral acquisition. Over the past decade, numerous broadly neutralizing ...IMPORTANCE: HIV-1 broadly neutralizing antibodies will be a key component of an effective HIV-1 vaccine, as they prevent viral acquisition. Over the past decade, numerous broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV. Despite an in-depth knowledge of their structures, epitopes, ontogenies, and, in a few rare cases, their maturation pathways during infection, bnAbs have, so far, not been elicited by vaccination. This necessitates the identification of key obstacles that prevent their elicitation by immunization and overcoming them. Here we examined whether CDRL1 shortening is a prerequisite for the broadly neutralizing potential of VRC01-class bnAbs, which bind within the CD4 receptor binding site of Env. Our findings indicate that CDRL1 shortening by itself is important but not sufficient for the acquisition of neutralization breadth, and suggest that particular combinations of amino acid mutations, not elicited so far by vaccination, are most likely required for the development of such a feature.
History
DepositionApr 18, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.3Nov 6, 2024Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: 426c.WITO.TM.SOSIP
C: 426c.WITO.TM.SOSIP
D: Fv domain of heavy chain of mAb 8-24
E: Fv domain of light chain of mAb 8-24
F: Fv domain of heavy chain of mAb 8-24
G: Fv domain of light chain of mAb 8-24
H: Fv domain of heavy chain of mAb 8-24
L: Fv domain of light chain of mAb 8-24
A: 426c.WITO.TM.SOSIP
hetero molecules


Theoretical massNumber of molelcules
Total (without water)376,89763
Polymers351,4369
Non-polymers25,46154
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 1 types, 3 molecules BCA

#1: Protein 426c.WITO.TM.SOSIP


Mass: 69535.422 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Cell line (production host): HEK 293E / Production host: Homo sapiens (human)

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Antibody , 2 types, 6 molecules DFHEGL

#2: Antibody Fv domain of heavy chain of mAb 8-24


Mass: 25560.518 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): HEK 293E / Production host: Homo sapiens (human)
#3: Antibody Fv domain of light chain of mAb 8-24


Mass: 22049.486 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): HEK 293E / Production host: Homo sapiens (human)

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Sugars , 5 types, 54 molecules

#4: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#5: Polysaccharide
alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#6: Polysaccharide
alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_d2-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#7: Polysaccharide alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#8: Sugar...
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 21
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of 426c.WITO.TM.SOSIP bound to three mAb 8-24 Fabs
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.21 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK 293E
Buffer solutionpH: 7.5
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.2particle selection
4cryoSPARCv4.2CTF correction
10cryoSPARCv4.2initial Euler assignment
11cryoSPARCv4.4.1final Euler assignment
13cryoSPARCv4.4.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2600000
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23004 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 263.39 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004320580
ELECTRON MICROSCOPYf_angle_d0.54227927
ELECTRON MICROSCOPYf_chiral_restr0.04153504
ELECTRON MICROSCOPYf_plane_restr0.00383357
ELECTRON MICROSCOPYf_dihedral_angle_d11.03457815

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